Bovine brain and pituitary fibroblast growth factors: comparison of their abilities to support the proliferation of human and bovine vascular endothelial cells

The mitogenic effects of brain and pituitary fibroblast growth factors (FGF) on vascular endothelial cells derived from either human umbilical vein or bovine aortic arch have been compared. Both brain and pituitary FGF are mitogenic for low density human umbilical endothelial (HUE) cell cultures maintained on either fibronectin- or laminin-coated dishes or on biomatrices produced by cultured cells such as bovine corneal endothelial cells or the teratocarcinoma cell line PF-HR-9. Pituitary FGF triggered the proliferation of HUE cells at concentrations as low as 0.25 ng/ml, with a half-maximal response at 0.55 ng/ml and optimal effect at 2.5 to 5 ng/ml. It was 50,000-fold more potent than commercial preparations of endothelial cell growth factor and 40 times more potent than commercial preparations of pituitary FGF. Similar results were observed when the effect of pituitary FGF was tested on low density cultures of adult bovine aortic endothelial cells. When the activity of brain and pituitary FGF on low density HUE cell cultures was compared, both mitogens were active. To confirm the presence in brain extract of both acidic and neutral, as well as of basic mitogen, for HUE cells, brain tissues were extracted at acidic (4.5), neutral (7.2), and basic (8.5) pH. The three types of extracts were equally potent in supporting the proliferation of either HUE or adult bovine aortic endothelial cells. When the various extracts were absorbed at pH 6.0 on a carboxymethyl Sephadex C-50 column, the neutral and basic extracts had an activity after adsorption similar to that of unadsorbed extracts. In contrast, extracts prepared at pH 4.5 lost 90-95% of their activity which was recovered in the adsorbed fraction containing FGF.     

Regulation of angiotensin converting enzyme activity in cultured human vascular endothelial cells

Angiotensin converting enzyme (ACE) of vascular endothelial cells is suggested to control vascular wall tonus through the conversion of angiotensin I (AI) to angiotensin II (AII) and the degradation of bradykinin. To obtain more insight into the pathophysiological significance of ACE of vascular endothelial cells, we studied the regulation of ACE produced by cultured human umbilical vein endothelial cells (EC). Phorbol 12-myristate 13-acetate (PMA) increased the cellular and medium ACE activity, accompanied by a marked morphological change in EC. N'-O'-dibutylyladenosine 3';5'-cyclic monophosphate (db-cAMP) increased only the cellular ACE activity and not the medium ACE activity. The effect of isoproterenol with 0.1mM theophylline mimicked that of db-cAMP. These findings suggest that PMA and cAMP-related agents participate in the control of vascular wall tonus through the positive regulation of ACE produced by vascular endothelial cells.     

Isolation of pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): partial chemical and biological characterization

Bovine pituitary fibroblast growth factor has been purified 222,000-fold to homogeneity by a combination of differential salt extraction, gel filtration, and ion exchange chromatography on Mono S column. Pituitary FGF is a single-chain polypeptide with an apparent molecular mass of 15,800 and an isoelectric point of 9.6. It is highly active in triggering the proliferation of bovine and human vascular endothelial cell [half-maximal stimulation at 23-40 pg/ml (1.5-2.6 pM) and saturation between 140 and 280 pg/ml (9.3-18.6 pM)]. It displays a similar activity on bovine vascular smooth muscle cells, corneal endothelial cells, granulosa and adrenal cortex cells, and rabbit costal chondrocytes.     

Immunoreactive fibroblast growth factor in cells of peritoneal exudate suggests its identity with macrophage-derived growth factor

Peritoneal exudate cells were collected from thioglycollate stimulated mice, extracted an examined for the presence of immunoreactive and bioactive fibroblast growth factor (FGF). The crude extract stimulated in a dose dependent fashion the proliferation of vascular endothelial cells derived from the bovine aortic arch. The extract also showed a parallel and dose-dependent inhibition of binding in a highly specific radioimmunoassay for FGF. The immunoreactive FGF (ir-FGF) contained in the extract was retained on a heparin-sepharose affinity column as is characteristic of pituitary FGF. Reverse-phase HPLC of the macrophage-derived material reveals one biologically active form of FGF which coelutes with the major form of immunoreactivity. The results demonstrate the presence of FGF in these cells and suggest that at least one of the hitherto unidentified mitotic activities in these extracts is due to a mitogen indistinguishable from FGF.     

Isolation and partial characterization of an endothelial cell growth factor from the bovine kidney: homology with basic fibroblast growth factor

Two kidney-derived mitogens have been isolated by ion exchange, heparin-Sepharose and reverse-phase high-performance liquid chromatography on the basis of their capacity to stimulate the proliferation of bovine vascular endothelial cells in vitro. Gas phase sequence analysis identified the amino terminal sequences His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-X-Lys-Asn-Gly-Gly-Phe-Phe-Leu and His-Phe-Lys-Asp-Pro-Lys-Arg-Leu, respectively. The sequences are identical to residues 16-32 and 16-23 of bovine basic pituitary Fibroblast Growth Factor (FGF). The possibility that these kidney-derived mitogens are related, if not identical, to pituitary basic FGF is supported by the observations that they have similar molecular weights (15-16 kDa), similar retention behavior on all steps of chromatography and similar amino acid compositions, and they share at least some structural homology. Moreover, the kidney-derived growth factors, like basic FGF, are potent stimulators of capillary endothelial cells, granulosa cells, adrenocortical cells and vascular smooth-muscle cells (ED50 = 50 pg). The results demonstrate the existence of a kidney-derived FGF and suggest that at least some of the mitogenic, angiogenic and neovascularising activities described to be present in the kidney are due to the presence of an FGF-like molecule in this tissue.     

Control of proliferation of bovine vascular endothelial cells.

The effects of Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF) on the proliferation of bovine vascular endothelial cells has been examined. FGF induces the initiation of DNA synthesis and cell proliferation in cloned endothelial cells of fetal and adult origin at concentrations as low as 1 ng/ml and is saturating at 50 ng/ml. EGF had no effect over the same range of concentrations. The mitogenic effect of FGF is blocked by a crude extract of cartilage. Platelet extract is also mitogenic for vascular endothelial cells although to a lesser extent than the purified FGF. In contrast to vascular endothelial cells, both EGF and FGF are mitogenic for vascular smooth muscle cells although EGF is less mitogenic than FGF at 100 ng/ml. The mitogenic effect of EGF and FGF on vascular smooth muscle is not blocked by the addition of a crude extract of cartilage, thus demonstrating the specificity of the chalone like effect of cartilage crude extract for endothelial cells.     

Paraquat depresses the activity of angiotensin converting enzyme expressed by human umbilical vein endothelial cells in culture

The activity of angiotensin converting enzyme (ACE) in cell lysate of cultured human umbilical vein endothelial cells (HUVEC) after a 24-hour incubation with 10(-3) and 10(-4)M of paraquat (PQ) was decreased. However, LDH released into the culture medium of HUVEC during the 24-hour incubation with PQ was not increased. Many investigators show that the change in serum ACE activity reflects the impairment of vascular endothelial cells. We showed in this report that ACE was decreased even at an early stage of endothelial injury induced by PQ, when LDH release is not yet increased.     

Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin

Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of being passage repeatedly while maintaining a high mitotic index. The stock cultures used for these studies have been passed weekly with a split ratio of 1 to 10 and are currently in their 30th passage. These cultures are indistinguishable from earlier passages when examined for the presence of Weibel-Palade bodies or Factor VIII antigen. We conclude that the use of FGF and thrombin can prevent the precocious senescence observed in most human endothelial cells cultures previously described.     

Bumetanide and furosemide inhibited vascular endothelial cell proliferation

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl–cotransport in the mitogenic signal of vascular endothelial cell proliferation. The activity of the Na+/K+/Cl– cotransport is dramatically decreased in quiescent subconfluent cells, as compared to subconfluent cells growing in the presence of FGF. The Na+/K+/Cl– cotransport activity of quiescent subconfluent cultures deprived of FGF decreased to 6%, whereas that of quiescent cells grown to confluency was reduced to only 33% of the activity of subconfluent cells growing in the presence of FGF. The basal low activity of Na+/K+/Cl– cotransport in the quiescent subconfluent vascular endothelial cells was dramatically stimulated by FGF. In order to explore the role of the Na+/K+/Cl– cotransport in the mitogenic signal of the endothelial cells, the effect of two specific inhibitors of the cotransport -furosemide and -bumetanide was tested on cell proliferation induced by FGF. Bumetanide and furosemide inhibited synchronized cell proliferation measured by direct counting of cells and by DNA synthesis. Inhibition by fuorsemide and bumetanide was reversible; removal of these compounds completely released the cells to proliferate. These results indicate that the effect of these drugs is specific and is not due to an indirect toxic effect. This study clearly demonstrates that the FGF-induced activation of the Na+/K+/Cl– cotransport plays a role in the mitogenic signal pathway of vascular endothelial cells. © 1994 Wiley-Liss, Inc.     

Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation

Survival and proliferation of many types of vascular endothelial cells are influenced by fibroblast growth factor (FGF)1. Removal of FGF from the medium of human umbilical vein endothelial cells (HUVEC) in culture resulted in death of the cells. Here we show that the death caused by deprivation of FGF is active death or apoptosis, and the process of apoptosis can be inhibited by cycloheximide, an inhibitor of protein synthesis. The present study shows apoptosis occurs in endothelial cells in culture. The process of active death of vascular endothelial cells is inhibited by growth factor. This mechanism may be important for the regulation of vascular organization through the degeneration of vessels.     

Role of protein kinase C in the inhibition by fibroblast growth factor of apoptosis in serum-depleted endothelial cells

Apoptosis in vascular endothelial cells is suppressed by fibroblast growth factor (FGF)1. In order to investigate the signal transduction system that regulates endothelial apoptosis, we studied the effects of several mitogenic factors. Apoptosis occurred in human vascular endothelial cells under serum-free conditions, and FGF inhibited apoptosis without a requirement of any cooperative factors, as distinct from the mitogenic response. Other mitogenic agents, such as epidermal growth factor, transferrin, transforming growth factor beta, and interleukin 1 etc., with the exception of dexamethasone, had no such inhibitory effects. The effect of FGF was mimicked by a phorbol ester and was prevented by an inhibitor of protein kinase C. The results suggest that the FGF and protein kinase C are important in endothelial apoptosis.     

Bovine fibroblast growth factor: comparison of brain and pituitary preparations

Bovine brain and pituitary fibroblast growth factors (FGF) have been compared with regard to their chemical and biological properties. Pituitary and one preparation of brain FGF (Prep A) contain a basic mitogenic activity, which migrates to the same position on electrophoresis in acid pH gels as detected by incorporation of [methyl- 3H]-thymidine into BALB/c 3T3 cells. In contrast, another preparation of brain FGF (Prep B) contains two mitogens, one (20-30%) indistinguishable from the basic components in pituitary and brain (Prep A) FGF preparations and an acidic activity (70-80%), pl 5-6, that migrates more slowly on acid gels, corresponding to the acidic component of brain FGF described previously (Thomas, K. A., M. C. Riley, S. K. Lemmon, N. C. Baglan, and R. A. Bradshaw. 1980. J. Biol. Chem. 255:5517-5520.) In agreement with that report, none of the mitogens comigrates with fragments of myelin basic protein. Pituitary FGF was virtually inactive, brain (Prep A) FGF had a small amount of activity, and brain (Prep B) FGF was highly potent (50% maximal stimulation at 15-30 ng/ml) in stimulating the growth of human umbilical vein endothelial (HUVE) cells. The acidic component of brain FGF, which is much more unstable at pH 8.5 than the basic one, can be protected by reducing agents, whereas the basic constituent of brain FGF as well as pituitary FGF is unaffected by reducing conditions. Thus, brain FGF preparations may contain two distinct mitogenic activities, one that is acidic and contains HUVE cell activity, and a basic mitogen that is similar to and may be identical with pituitary FGF.     

A unique family of endothelial cell polypeptide mitogens: the antigenic and receptor cross-reactivity of bovine endothelial cell growth factor, brain-derived acidic fibroblast growth factor, and eye-derived growth factor-II

Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [125I]ECGF were performed to determine the cross-reactivity of ECGF with bovine acidic pI brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II [EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [125I] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGF-II is potentiated by the glycosaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF-II belong to a common family of polypeptide growth factors.     

Maintenance of vascular integrity in the embryo requires signaling through the fibroblast growth factor receptor

Basic fibroblast growth factor (FGF)-2 is important for vessel formation and/or maintenance of vascular integrity in the embryo. FGF signaling may be mediated through transmembrane tyrosine kinase receptors or directly through intracellular pathways that do not involve receptor activation. To determine the role of receptor-mediated signaling in endothelial cells, an adenovirus encoding truncated FGF receptor (FGFR)-1, under the control of the cytomegalovirus promoter, was expressed in endothelial cells. FGF signaling was impaired, as indicated by inhibition of MAPK phosphorylation. Functional consequences included inhibition of endothelial cell migration and induction of apoptosis. To address the role of endothelial FGFR signaling in vascular development, recombinant adenovirus encoding a dominant-negative FGFR was injected into the sinus venosus of embryonic day 9.0 cultured mouse embryos. Previous studies demonstrated that transgenes delivered via adenovirus, under the control of the cytomegalovirus promoter, are expressed selectively in the developing vasculature. Embryos expressing a control adenovirus developed normally, whereas those expressing the FGFR-1 mutant exhibited abnormal embryonic and extra-embryonic vascular development. These data demonstrate that FGF, by signaling through the FGFR, plays a pivotal role in the development and maintenance of a mature vascular network in the embryo.     

Elabela prevents angiotensin II-induced apoptosis and inflammation in rat aortic adventitial fibroblasts via the activation of FGF21–ACE2 signaling

Apoptosis, inflammation, and fibrosis contribute to vascular remodeling and injury. Elabela (ELA) serves as a crucial regulator to maintain vascular function and has been implicated in the pathogenesis of hypertensive vascular remodeling. This study aims to explore regulatory roles and underlying mechanisms of ELA in rat aortic adventitial fibroblasts (AFs) in response to angiotensin II (ATII). In cultured AFs, exposure to ATII resulted in marked decreases in mRNA and protein levels of ELA, fibroblast growth factor 21 (FGF21), and angiotensin-converting enzyme 2 (ACE2) as well as increases in apoptosis, inflammation, oxidative stress, and cellular migration, which were partially blocked by the exogenous replenishment of ELA and recombinant FGF21, respectively. Moreover, treatment with ELA strikingly reversed ATII-mediated the loss of FGF21 and ACE2 levels in rat aortic AFs. FGF21 knockdown with small interfering RNA (siRNA) significantly counterbalanced protective effects of ELA on ATII-mediated the promotion of cell migration, apoptosis, inflammatory, and oxidative injury in rat aortic AFs. More importantly, pretreatment with recombinant FGF21 strikingly inhibited ATII-mediated the loss of ACE2 and the augmentation of cell apoptosis, oxidative stress, and inflammatory injury in rat aortic AFs, which were partially prevented by the knockdown of ACE2 with siRNA. In summary, ELA exerts its anti-apoptotic, anti-inflammatory, and anti-oxidant effects in rat aortic AFs via activation of the FGF21–ACE2 signaling. ELA may represent a potential candidate to predict vascular damage and targeting the FGF21–ACE2 signaling may be a promising therapeutic intervention for vascular adventitial remodeling and related disorders.

     

Aortic endothelial cells synthesize basic fibroblast growth factor which remains cell associated and platelet-derived growth factor-like protein which is secreted

Cultured bovine aortic endothelial cells synthesize growth factors which markedly differ in the regulation of their storage and secretion. Endothelial cell lysates, but not conditioned medium, contain a growth factor activity that appears to be basic fibroblast growth factor (FGF) by the following criteria: (1) it elutes from heparin-Sepharose at 1.4-1.6 M NaCl; (2) it is mitogenic for bovine aortic and capillary endothelial cells; (3) it is heat sensitive but stable to dithiothreitol; (4) it has a molecular weight of about 18,000 daltons; and (5) it cross-reacts with antiserum directed against basic FGF. In contrast, endothelial cell conditioned medium, but not lysates, contains a growth factor activity that (1) elutes from heparin-Sepharose at 0.4-0.5 M NaCl; (2) is mitogenic for fibroblasts and vascular smooth muscle cells but not for capillary endothelial cells; (3) is heat stable and dithiothreitol sensitive; and (4) competes with platelet-derived growth factor (PDGF) for binding to fibroblasts. From these criteria, it appears that endothelial cells secrete into the medium growth factors some of which are PDGF-like, but secrete little if any basic FGF. It is suggested that endothelial cell-associated basic FGF acts in an autocrine fashion to stimulate endothelial cell proliferation in response to endothelial cell perturbation or injury. On the other hand, the endothelial cell-secreted growth factors which are smooth muscle cell but not endothelial cell mitogens might exert a paracrine function on neighboring cells of the vessel wall.     

Conditioned medium from endothelial cell cultures can restore the normal phenotypic expression of vascular endothelium maintained in vitro in the absence of fibroblast growth factor

Bovine vascular endothelial cells continuously maintained and grown in the presence of FGF adopt at confluence the configuration of a cell monolayer composed of contact-inhibited cells which do not overgrow each other and which are highly flattened and closely apposed. Such cultures exhibit structural and morphological characteristics similar to those observed with their in vivo counterparts. These include the production of an extracellular matrix consisting mostly of basement membrane collagen and fibronectin localized exclusively beneath the cell monolayer, but not on top of it, as well as a nonthrombogenic, blood-compatible apical cell surface. Removal of fibroblast growth factor (FGF) from adult bovine aortic endothelial cell (ABAE) cultures results within three passages in the loss by the cells of their characteristic contact-inhibited morphology. The cells, which during their logarithmic growth phase divide with a greatly increased doubling time, become larger and more elongated. Confluent cultures, instead of adopting the morphology of a contact inhibited cell monolayer, are now composed of overgrowing cells. Parallel with the morphological alterations taking place within the culture, the cells also lose the polarity of cell surfaces characteristics of the vascular endothelium. Formation of an extracellular matrix composed primarily of fibronectin and collagen types I, III, and IV is observed on both the apical and basal cell surfaces. Platelets which previously did not bind to the apical cell surface now become capable of binding to it. CSP-60, a major cell surface protein present in highly confluent and contact-inhibited vascular endothelial cell cultures, can no longer be detected. Exposure of confluent endothelial cell cultures, maintained in the absence of FGF to medium conditioned by cells which had been grown in the presence of FGF, but maintained in its absence upon reaching confluence led, within four to eight days, to a reversion of the altered phenotype. This medium has little or no mitogenic activity and retains a full activity in the absence of serum or after depletion of its fibronectin content by affinity chromatography on a gelatin-Sepharose column. Cultures which were previously composed of cells growing in multiple layers reorganized into a single cell monolayer composed of closely apposed and highly flattened cells. The cultures thereby regained the contact-inhibited morphology characteristic of the vascular endothelium. Concomitant with this cellular reorganization, the extracellular matrix disappeared from the apical cell surface, the cells regained their nonthrombogenic properties, and CSP-60 reappeared as one of the major cell surface proteins. These results suggest that vascular endothelial cells secrete a soluble factor(s) which can restore the normal morphology and function lost following removal of FGF from the medium. Such a factor(s) may be involved in maintaining the differentiated state of the vascular endothelium.     

Fibroblast growth factor 16 and 18 are expressed in human cardiovascular tissues and induce on endothelial cells migration but not proliferation

Endothelial cells line the blood vessel and precursor endothelial cells appear to have a pivotal effect on the organ formation of the heart, the embryonic development of the kidney, and the liver. Several growth factors including the fibroblast growth factors (FGF) seem to be involved in these processes. Ligands such as basic FGF produced and secreted by endothelial cells may also coordinate cellular migration, differentiation, and proliferation under pathological conditions including wound healing, tumorgenesis, and fibrogenesis in the adult. Recently we demonstrated the expression of two secreted FGFs, FGF16, and FGF18, in HUVEC and in rat aortic tissue. In the present report, we confirmed by RT-PCR analysis that FGF18 is wildly expressed in the cardiovascular tissue, while FGF16 showed a more restricted expression pattern. HUVEC clearly demonstrated chemotaxis towards FGF16 and FGF18. Both FGFs also enhanced cell migration in response to mechanical damage. However, recombinant FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in a three-dimensional in vitro angiogenesis assay. Fgf18 expression was earlier reported in the liver, and we detected FGF18 expression in liver vascular and liver sinusoidal endothelial cells (LSECs), but not in hepatic parenchymal cells. Recombinant FGF18 stimulated DNA synthesis in primary hepatocytes, suggesting, that endothelial FGF18 might have a paracrine function in promoting growth of the parenchymal tissue. Interestingly, FGF2, which is mitogenic on endothelial cells and hepatocytes stimulates a sustained MAPK activation in both cell types, while FGF18 causes a short transient activation of the MAPK pathway in endothelial cells but a sustained activation in hepatocytes. Therefore, the difference in the time course of MAPK activation by the different FGFs appears to be the cause for the different cellular responses.     

Tissue-Specific Expression of Transgenic Secreted ACE in Vasculature Can Restore Normal Kidney Functions,but Not Blood Pressure,of Ace-/- Mice

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.     

Dual activities of 22-24 kDA basic fibroblast growth factor: inhibition of migration and stimulation of proliferation

Basic fibroblast growth factor (FGF2) is synthesized as four isoforms with molecular weights of 24, 22.5, 22, and 18 kDa, with each of the three higher molecular weight forms (hmwFGF2) produced by the initiation of translation at one of three upstream CUG codons. We have shown that bovine arterial endothelial cells export the high molecular weight forms of FGF2 (hmwFGF2) in a 17beta-estradiol-dependent manner (Piotrowicz et al., 1997, J Biol Chem 272:7042-7047). To determine whether the hmwFGF2 forms affected cell behavior after release, we evaluated the effect of recombinant hmwFGF2 on the growth and migration of endothelial cells and mammary carcinoma cells (MCF-7). Treatment with the recombinant protein resulted in the inhibition of endothelial cell migration by 45% and MCF-7 cell migration by 70%. HmwFGF2-dependent inhibition was observed when endothelial cell migration was stimulated by 18 kDa FGF2 or vascular endothelial growth, and MCF cell migration was stimulated with insulin-like growth factor. In each case, inclusion of an antibody against the 55 amino acid amino terminal end of 24 kDa FGF2 abrogated the inhibition of migration, while antibodies to the 18 kDa FGF2 domain had no effect. When endothelial cells were cultured under conditions which promoted export of hmwFGF2, a 40% decrease in motility was observed which was reversed by the antibodies to the 24 kDa FGF2. Thus, both recombinant and endogenously produced hmw-FGF2 are capable of inhibiting migration. In contrast to the ubiquitous effect on migration, hmwFGF2 had no effect on endothelial cell growth but stimulated MCF-7 growth equally as well as the 18 kDa FGF2 (threefold). Antibodies to the 18 kDa domain of 24 kDa FGF2 blocked the growth-promoting activity of hmwFGF2, but those to the amino terminal end were ineffective. These data suggest that hmwFGF2 has dual activities, an inhibitory effect on cell migration and a growth-stimulating effect. The two activities can be localized to different parts of hmwFGF2: inhibitory activity to the amino terminal 55 amino acids (which are absent from the 18 kDa FGF2) and growth-promoting activity to the 18 kDa domain. Therefore, the ratio of hmwFGF2 and 18 kDa FGF2 in the extracellular space may provide a mechanism of control for angiogenesis and mammary tumor development.